Genetic Approaches to the Control 
of Mycobacterial Disease 
William R. Jacobs, Jr., Ph.D. — Assistant Investigator 
Dr. Jacobs is also Assistant Professor in the Departments of Microbiology and Immunology and of Molec- 
ular Genetics at Albert Einstein College of Medicine. He received a B.A. degree in mathematics at Edinboro 
University of Pennsylvania and a Ph.D. degree in molecular cell biology from the University of Alabama 
at Birmingham. His doctoral work on Mycobacterium leprae was performed in the laboratory of Josephine 
Clark- Curtiss and Roy Curtiss III. His postdoctoral studies with Barry Bloom focused on developing genetic 
systems for the mycobacteria. 
TUBERCULOSIS, caused by Mycobacterium 
tuberculosis, continues to be the major 
cause of death throughout the world today. The 
World Health Organization estimates that each 
year there are approximately 10 million new 
cases of tuberculosis and over 3 million deaths. 
After 32 years of a steadily decreasing incidence 
in the United States, a surprising and alarming 
increase in the numbers of new cases has been 
reported in many of our cities in the last four 
years. This is thought to be a result of the AIDS 
(acquired immune deficiency syndrome) epi- 
demic and is of considerable concern. Tubercu- 
losis is not only a disease common to AIDS pa- 
tients but is also one of the few diseases that can 
be readily spread from persons with AIDS to the 
general population. 
Another microorganism of the same genus, My- 
cobacterium avium, although not a pathogen to 
healthy individuals, is a major opportunistic 
pathogen in AIDS. Mycobacterium leprae is the 
causative agent of leprosy, an affliction dating 
back to ancient times that affects over 1 3 million 
people in the world today. 
In contrast to the pathogens, BCG (bacille Cal- 
mette-Guerin) , the tuberculosis vaccine, has 
been used through parts of the world since 1922 
to prevent tuberculosis. We believe that BCG rep- 
resents an ideal candidate as a recombinant vac- 
cine vector containing foreign antigen genes, be- 
cause it is safe, has excellent adjuvant properties, 
and should elicit long-lasting immunity. Thus the 
goals of my laboratory are 1) to dissect patho- 
genic mycobacteria genetically in order to de- 
velop effective strategies to cure and prevent my- 
cobacterial infections and 2) to engineer BCG as 
a recombinant vaccine vector. 
Historically mycobacteria have played a promi- 
nent role in the development of microbiology. 
Robert Koch, in 1882, established the criteria 
(Koch's postulates) by which one ascertains 
whether an organism causes an infectious disease 
when he established that the tubercle bacillus 
causes tuberculosis. By analogy, "Koch's molecu- 
lar postulates" is the method by which one ascer- 
tains that a characteristic of a bacterium, such as 
its virulence, is caused by a particular gene. The 
essential steps involve 1 ) identification of a mu- 
tant of the bacterium that lacks some characteris- 
tic, 2) cloning of an individual gene(s) from the 
parent bacterium, and 3) transfer of the cloned 
normal genes into the bacterial mutant to demon- 
strate restoration of the original characteristic. 
For example, to identify a gene required for viru- 
lence of M. tuberculosis, we would 1) identify 
an avirulent variant of the organism, 2) clone the 
genes from the virulent strain, and 3) introduce 
the putative virulence gene back into the aviru- 
lent mutant to demonstrate that this gene confers 
virulence. 
For the mycobacteria, these sorts of experi- 
ments entail considerable difficulties. The organ- 
isms have been objects of study since Koch's pio- 
neering work, but their genetic analyses had not 
been achieved, primarily because of their slow 
growth. The tubercle bacillus, which multiplies 
only once every 24 hours, requires three weeks 
to form a colony from a single cell. In contrast, 
Escherichia coli yield visible colonies in eight 
hours. The leprosy bacillus has yet to be culti- 
vated in the laboratory and can only be grown in 
mouse footpads or the nine-banded armadillo. 
Furthermore, although recombinant DNA tech- 
nology has enabled us to clone any mycobacterial 
gene, the technologies to transfer recombinant 
DNA back into mycobacteria did not exist five 
years ago. 
We took advantage of bacterial viruses that in- 
fect mycobacteria, called mycobacteriophages, 
as the building blocks for developing vectors ca- 
pable of transferring recombinant DNA effi- 
ciently into mycobacterial cells. A novel hybrid 
vector, called a shuttle phasmid, was con- 
structed. It replicates in mycobacteria as a phage 
and in E. coli as a plasmid, which permitted us to 
introduce cloned genes into a wide variety of my- 
cobacterial strains for the first time. 
Using these vectors, we identified selectable 
marker genes that have allowed us to develop 
phage- and plasmid-derived vectors, as well as 
systems to insert genes into the mycobacterial 
chromosomes. We have isolated mutants of a fast- 
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